Art of growing and distributing nitro-gathering bacteria.



PATENTED SEPT. .10, 1907! G. H. HARP-THOMAS. v ART OF GROWING ANDDISTRIBUTING NITRO GATHERING BACTERIA.

' APPLICATION FILED JAN, 17, 1907.

Wilma/n20 UNITED: STATES? OFFICE,

GEORGE HERBERT mar-moms, QETQRONTQ'oNTARIO, CANADA.

-ART or' -enowme am) ms rnrsormo mac-surname Bac'rnnn.

Specification-of latter-a Patent.

Patented Sept. 10, 1907.

- To all whom it may Be it known that I, Gnonen Hnnnnn'r Emu- THOMAS, acitizen of New Zealand, residing at 400 Bloor street, Toronto, Canada,have invented certain new and useful Improvements in the Art or Growingand Distributing Nitrogen-Gathering Bacteria; and I others skilled inthe art to which itappertains to make also known that it isadvantageousto grow cultures of such nitrogen gathering-bacteria .in laboratoriesand distribute the same ag'riculturists in order that th'ey maydirectlyinoc' ate the seeds before-planting,

whereby an increased growth of the plants and an increased yield isobtained. In growing such cultures in the laboratory it has-been thepractice heretofore first to obtain someof the desired bacteria fromplants talgenfrom the field or. garden andthen to implant such bacteria.so obtained upon a suitable mediiirn which is then keptunder the desiredconditions to propagate the colonies, the culture thus obtained beinggenerally known as the stock culture, which labora tory stock cultureserves as a.source of germs from which to propagate the cultures to. bedistributed to the to the growing plant for the germs, but that from thestock culture other cultures might be propagated indefinitely. However,owing to the fact that the characteristic properties of these bacteriabecome modified by the food upon which they live, it results that alaboratory stock culture,after some time, consists'of bacteria whichhave, to a certain extent, lost their power of fixing nitrogen for theplant, or in other words have become what is known as attenuated, thatis to say,

- less virulent than before and therefore less valuable to abovedescribed disadvantage, andjthis I do by the means more fully explainedhereinafter.

A licant liladilannsry 17.19107. smunaaoa'res.

with the. old method, which is due principallyto the necessity for thefrequent renewal of the stock culture and to the great'similarity whichexists between vantageiis that when a new culturehas been to be thedesired nitrogen gathering bacteria and yet test of a culture is to putitin practical use, that is, to inoculate seed with it and then grow aplant from such under such conditions as often to render the testInconclusive, for the reason that the possibility of the inoculation ofthe plant trom sources other than the stock culture, was not absolutelyexcluded. Consequently, by the old method ol'procedure it is possible tohave cultures of bacteria satisfactorily pass all the tests to farmer asnitrogen-gathering bacteria, when in fact they were not nitrogengathering bacteria but were merely some other form of bacteria whichwere useless for the purpose intended.

It is a further object of my invention to avoid this explainedFurthermore, although the nitrogen gathering bacteria so far generallyknown are supposed to be and probably are of one species only, theresults attained in actual practice have shown that there is adifference in the bacteria dependent upon the species of plant upon(which-the stock culture was obtained. For example, bacteria obtainedoriginally from the roots of red clover will not produce as satisfactoryresults when employed for inoculating alsike as would bacteriaoriginally obtained from the roots of an alsike plant, and on the otherhand the latter will not be satisfactory for the inoculation of re dclover. Ebparently the characteristies of the bacteria are modified bythe food upon which they live, so that when for example, the red cloverbacteria are placed upon red clover seed they are ready to thrive on therootsiof the growing plant, whereas when placed upon some other seed, asfor example, on alsikeseed, they find their environment and food lesssuitable to them and hence a are-not as vigorous in their attacks on theplant roots. However, the bacteria have the power of finally adaptingthemselves to the changed conditions, that is to say, after manygenerations are produced uponthe new kind of hosttherace finally becomesaccustomed to the new host and is enabled to carry on its tion of thebacteria. to its strange host requires some time, and the consequence isthat seed inoculated .seed. This test has been employed heretofore, but

which they have been submitted and be sent to the disadvantage,whichl doby the means hereinafter function satisfactorily, Unfortunately, thisadapts There is also a further great disadvantage connected thenitrogengathering bacteria and other soil bacteria which are useless forinoculating seed} This disadit be examined under the microscope, it mayappear really may be some form of soil-bacteriausele'ss for the purposerequired. Apparently the onlyreally reliable.

nation. Owing to this necessity .for employing the the receptacle A. Asa filter a plug of cotton is genlamp, in order to make certain that allorganisms on with bacteria unadapted to it does not have the ad Ivantage of a quick growth when first starting and thus a part of thegrowing season is lost, which would not be the case'were the proper kindor variety -of bacteria present on the seed at the moment of germiproperkind of bacteria for each kind of plant, it has been necessary tomaintain many different kinds of stock cultures in the laboratory eachcontaining bacteria adapted for a particular plant. Also it has beennecessary first to ascertain from a purchaser, the kind of plant whichhe intended to inoculate before his order could be filled. And as it isnecessary to make up the cultures in distributing packages prior to theopening-of the season for which they are to be used, so that orders maybe filled without delay, it has been necessary to make a supply ofdistributing packages of each kind of bacteria.

It is a further object of my inv ention to avoid this difliculty which Ido by the means hereinafter described.

With these general objects in view, and some others which will beobvious to those skilled in the art from the description hereinafter, myinvention consists in the features and details which will first bedescribed in connection with the accompanying drawing and thenparticularly pointed out in the claims.

The drawing is an elevation of a stock culture embodying one part of myinvention.

Referring to the drawing, A is a receptacle or container, in this caseof suitable transparent material, as for example, glass, and providedwith an opening at one side arranged to be closed by a suitable closure.Inthe present example, the desired result is attained by providing thereceptacle with a tubulature indicated at a which maybe closed by astopper which will exclude germs, as for example, a tightly fittingglass stopper B.

The upper part of the receptacle A is provided with means for supplyingit with sterilized air that is to say air which is free from germs ofany kind. In the present instance, I obtain this result in a simplemanner by providing the receptacle A at the upper part with an opening,.a in which may be inserted a filtering device for filtering the air sothat all germs will be removed as the outside air passes through thefilter into erally employed, this being indicated at 0.

Within the receptacle, I place a suitable medium, taking care to haveboth sterilized by any well known methods, the medium in the preferredform of my invention being non-nitrogenous and substantiallytransparent. In my Patent 816,850, dated April 3,

1906, I have described a medium which is very suitable for this purpose.In this medium, I place one or more seeds taken from the desired kind ofplant in such a way as to insure that the seed is free from bacteria ofany kind. For the purpose of accomplishing this result I may proceed inthe following manner. I take a seed-head of the desired plant andcarefully sterilize the same by passing it several times through theflame, such as the flame of an alcohol the outside of the seed-head aredestroyed by the heat. The seeds themselves within their seed cases orpods are not injured by the heat if the work is properly done.Apparently there are noinjuriousgerms within the seed-pod, if the latteris perfect,

in a suitable situation in order to receive a proper amount of light andheat, whereby the seed germinates and the bacteria at once start to workin the rootlets. v

The medium being non-nitrogenous, the bacteria are compelled to obtaintheir entife supply of nitrogen from the atmosphere. The plant in turntakes its nitrogen from the bacteria and thus grows rapidly, a littlesterilized water being added to the medium from time to time as may berequired. As the bacteria are growing and multiplying upon their naturallidst, they have a vitality and obtain a virulency which is even greaterthan that of the bacteria living upon a plant growing under naturalconditions in earth. Furthermore, as the medium is transparent thedevelopment of the colonies may be watched and the growth of the plantitself serves to determine whether the bacteria in the medium are thetrue nitrogen gathering bacteria or not for if they are not the plantwill not thrive, whereas if they are the plant -will flourish and willdevelop tubercles on the roots.

Such a laboratory stock culture being once established there will be noneed thereafter to goto the original source for bacteria, namely, to thesoil grown plant, because there is not only no loss in virulency of thebacteria on a stock culture such as I have described, but if anythingan. increase above the average of those cultures made directly from thesoil-grown plant. When now it is desired to make other cultures from thedescribed stock culture it is only necessary to remove the stopper fromthe tubulature, insert a sterilized pair of forceps and remove atubercle from the roots of the plant which may then be used in anydesired manner to inoculate the new medium. The

bacteria thus obtained are in a most virulent form.

tubercle ior inoculating a fresh medium from the stock culture, it isalso possible to use some other portion of the root of the plant, as forexample, a piece of one of the small root hairs, for these also containsome bacteria.

While I have hereinabove described the receptacle and its contents as astock culture, it is also to be understood that when a stock culture isobtained by V the old method, my invention may be employed as a meansfor testing such stock culture. That is, the seed may be planted in themedium in the receptacle as hereinbe'iore I p I t x to serve as astockciilture; but merely to determine bed then inoculated not whetheror not the bacteria employed are of -,the

proper kind. For such a purpose the receptacle may be smaller, becauseit is not necessary to provide much room for the .plant.. The firstgrowth of the" as transparent as possible and the receptacle ma y besmall enough so that it can conveniently be examined under themicroscope. I have found an ordinary test tube with a plug of cotton inits open end to serve sat-- isfactorily. With suchtest cultures it ispossible to observe the bacteria at work upon the plant. This test isparticularly useful in exposing to-view the 'nitrogen gatherers whichenter the roots but for some growing therein may be prepared and kept onhand.

The distributing vessels may be prepared and filled with medium,inoculated and sealed in the manner should be poured on top of themedium and the rece'ptacle then closed, as for example, by hermeticallysealing the same, whereupon thedistributingpackage is ready for shipmentor storage. t

The advantage of the solid medium is thatthere is less danger ofspilling the same in handling the vessels prior to sealing and henceless care is required in placing them in the incubator. But the foremostadvantage is that the growth of the colonies may be observed with asolid medium whereas it cannot withaliquid medium. The advantage of theliquid medium is that the bacteria can subdivide more readily than in asemi-solid medium, and hence will propagate quicker.

fore, by following the process set forth above, I attain the advantageof a 'solid medium during the period of preparation of the culturesprior to sealing and yet also attain the advantage of the liquid mediumafter sealing, thus leaving the bacteria in a favorable condition for a.

further development during storage and shipment.

In the above description, I have assumed that the medium in'thedistributing vessels is inoculated with one kind only of. the nitrogengathering bacteria; However, an important feature of my inventionconsists in inoculating the medium in each distributing vessel with aplurality of kinds of nitrogen gathering bacteria, as will now be morefully explained. To do this, I

Thereemel with cultures taken separately from the differ- 'e'rit stockcultures, thus combining in one culture bac teria suitable for differentkinds of plants. After this composite stock culturehas 'been formed, thedistributing vessels are inoculated from it whereby instead of eachdistributing vessel containing only one kind or variety of the nitrogengathering bacteria, it contains a plurality of kinds. Consequently, sucha culture is suitable for use in inoculating the seed of anyone of thekinds of plant from which the bacteria were originally obtained.However, and this is an important feature of my invention, it is notnecessary to use as much if any larger quantity of such a compositeculture in inoculating the seed than is required with the simpleculture, for the reason that there is enough of the specific kind ofbacteria to' attack the rootlets at the time the seed germinates andthus start the young plant into rapid growth by which time the otherkinds of bacteria have adapted themselves to their changed conditionsand then can thrive on the plant roots and assist in the 'growth of'suchplant A further advantage of this plan is that it gives a means ofgraduating the intensity of the attack on the young rootlets of thegerminating seed while at the same, time, insuring the presence ofsuflicient bacteria to properly stimulate the growth of the plant afterit has arrived at a more vigorous stage.

From laboratory experiments, I have ascertained that it is possible toinjure instead of aid the germinating plant by the employment oftoo-large a colony of virulent bacteria during the germinating of theseed.

In fact, in experiments carried out by me with peas,

the bacteria had not only retarded the growth of the rootlets but hadactually reduced the pea seed to a.

inere husk or shell. All danger from this source is entirely avoided bythe use of the composite culture.

- Having thus described my invention, what I claim a new and desire tosecure by Letters Patent, is:

1. The process of developing a bacteriological culture.

which conslsts 1n growing-in a medium a plant inoculated with thedeslred bacteria whlle excludlng all contaminatlons-from said plant andmedium.

.2. The process of developing a bacteriologlcal culture, which consistsin growing In a non-nitrogenous medium.- a plant lnoculated withnitrogen gathering bacteria while excluding all contaminations 'fromsaid plant and medium.

3. The process of developing a bacteriological culture, which consistsin growing a plant, inoculated with the desired bacteria within a germproof lnclosure.

4. The process of developing a bacterlologlcal culture. which consistsin growing a plant inoculated with the deslred bacteria, within a germproof lnclo'sure, while maintaining a restricted supply of sterilizedair to sald lnclosure.

5. The process of developing a bacterlologlcal culture.

which consists in growing a plant inoculated with the desired bacteriawithin a transparent germ proof lnclosure. 6. The process of developinga bacteriological culture. which consists in growing, in a substantiallytransparent medium, a plant inoculated with the desired bacteria whileexcluding all contamlnatlons from the plant and medium by a transparentgerm proof incloshi'e.

, 7. The process of developing a bacteriological culture, whlch conslstsin plantlng, in a sterilized medium, wlthln a germ proot lnclosure, avital seed free from contarnlnatlng germs maintaining a supply ofsterilized air to said inclosure and supplying sterilized water to theplant.

The process of developing a bacteriological which consists in growing aplant'lnoculated the desired bacteria, within a germ proof lnclosure,and then i). The process of developing a bacteriological culture,-

10. The process of developing a bacteriological culture,

which consists in growing a plant, inoculated with the desired bacteria,within a germ proof inclosure, removing a'ncdule from said plantand-inoculating a medium with.

the same.

11. The'process .which consists in preparing a composite cultureconsisting of a plurality of kinds of nitrogen gathering bacteria, theninoculating seed with such composite culture and planting the seed.

12. The-process which consists in inoculating seed with a compositeculture consisting of a plurality of kinds of nitrogen gatheringbacteria, one of whichkinds is specific to said seed, and then plantingthe seed.

13. The process which consists in inoculating the medium with aplurality of different kinds of nitrogen gathering bacteria to form acomposite culture, and then preparing from said composite culturethe'cultures to be distributed.

14. The process of producing a bacteriological culture, which consistsin growing a culture on a substantially solid medium and then adding aliquid to the solid'medium.

15. The process of producing a bacteriological culture, which consistsin first preparing a substantially solid medium, in a distributingvessel, inoculating it with nitrogen gathering bacteria, maintaining themedium under the required condition to develop the culture, then addingwater to the top of the medium and finally sealing said distributingvessel 16. A bacteriological culture comprising a medium, a plantgrowing in fsaid medium and inoculated with the desired bacteria, incombination with a germ proof re ceptacle inclosing the plant andmedium.

17. The combination, with a transparent germ prooif receptacle, of amedium within said receptacle, and a plant inclosed by said receptacleand rooted in the medium, said plant being inoculated with the desiredbacteria.

18. The combination, with a germ proof receptacle'provided with meansfor maintaining a supply of sterilized air to its int rlor, of a mediumwithin said receptacle, and a plant inoculated with the desired bacteriainclosed by said receptacle and rooted in the medium. v 19. Thecombination, with a germ-proof receptacle pervious to air, of a mediumwithin said receptacle, and a plant inoculated with the desired bacteriainclosed by said receptacle and rooted in the medium.

20. The combination, with a germ-proof transparent receptacle, of asubstantially transparent medium within said receptacle and a plantinoculated with the desired bacteria lnclosed and protected againstcontaminations by said receptacle and rooted in the medium.

21. The ,combination, with a glass vessel having an air inlet and an airfilter in said inlet, of a medium in said vessel, and a plant inoculatedwith the desired bacteria inclosed by the vessel and rooted in themedium.

22. The combination, with a vessel, having an air inlet and a lateralopening, a closure for said opening and an air filter in the air inlet,a medium in said vessel and accessible through the lateral opening and aplant inoculated with the desired bacteria, inclosed by the vessel androoted in the medium. Y

23. A bacteriological culture comprising a plant inoculated with thedesired bacteria, in combination .with a germ-proof receptaclesurrounding said plant and provided with means for permitting access tothe roots of said plant:

24. A bacteriological culture comprising a medium, a plant growing .insaid medium and inoculated with the desired bacteria, in combinationwith a germ-proof receptacle inclosing the plant and medium and providedwith means for-permitting access tothe roots of said plant.

' 25. A distributing package of nitrogen gathering bacteria containing aplurality of kinds of such bacteria.

26. A distributing package of nitrogen gathering bacteria comprising acomposite culture of several kinds of virulent nitrogen gatheringbacteria.

27. A distributing package comprising-a sealed receptacle containingasubstantially solid and also a liquid medium, and nitrogen gatheringbacteria in said media.

28. A distributing package comprising a receptacle,

means for restricting the access of air to said receptacle,

bacteria inclosed by said receptacle and rooted in the medium. 7

31. A bacteriological culture comprising a nitrogen-free medium, a plantgrowing in said-medium and inoculated with nitrogen gathering bacteria,in combination with 'a germ-proof receptacle, inclosing the plant andmedium,

. said receptacle being provided with means for maintaining a supply ofsterilized air to its interior and with means for permitting access tothe roots of said plant.

32. The process of developing a bacteriological culture,

which consists in growing in a non-nitrogenous medium,

a plant inoculated with nitrogen-gathering bacteria within a germ-proofinclosure, while maintaining a restricted supply of sterilized-air tothe interior of said inclosure, removing a nodule from said plant andinoculatinga medium with said nodule.

33. The process of producing a bacteriological culture, which consistsin first preparing a substantially solid medium, in a distributingvessel, inoculating it with nitrogen gathering bacteria, maintaining themedium under the required condition to develop the culture, then addingliquid to the top of the medium and finally sealing said distributingvessel.

34. A distributing package comprising a receptacle, 9. medium withinsaid receptacle and a plurality of kinds of bacteria in said mediuin,one of said kinds of bacteria being a nitrogen-gathering bacteriaspecific to the particular plant on-which the culture is to be used.

In testimony whereof I hereunto aflix my signature in the presence oftwo witnesses.

GEORGE HERBERT EARP-THOMAS. Witnesses M. C. MASSIE, E. O. HILDEBRAND.

